Comparative Proteomics Kit I:
Protein Profiler Module
Bio-Rad 166-2700EDU
32 students
List Price: $203.75 Refill: $ 94.00
Comparative Proteomics Kit I: Protein
Profiler Module
Laemmli sample buffer
Kaleidoscope™ prestained standards
Tris-glycine-SDS electrophoresis buffer
Bio-Safe™ Coomassie stain for proteins
Actin and myosin standard
Dithiothreitol (DTT)
Pipet tips for gel loading
Test tubes,transfer pipets, gel-staining trays, test tube
holders
Teacher's Guide, Student Manual, and graphic Quick
Guide 1
Required Accessories Not Included in Kit
Fish samples 5–8 types
Adjustable micropipets, 2–20 µl
Power supplies
Water bath
If using polyacrylamide gel electrophoresis:
Vertical gel electrophoresis chambers
Precast polyacrylamide gels
Is There Something Fishy About Teaching
Evolution?
Explore Biochemical Evidence for
Evolution
• Analyze protein profiles from a variety of fish
• Study protein structure/function
• Use polyacrylamide electrophoresis to separate proteins by size
• Construct cladograms using data from students’ gel analysis
• Compare biochemical and phylogenetic relationships.
• Sufficient materials for 8 student workstations
•
Can be completed in three 45 minute lab sessions
Workshop Timeline
• Introduction
• Sample Preparation
• Load and electrophorese protein samples
• Compare protein profiles
• Construct cladograms
Can biomolecular evidence be used to
determine evolutionary relationships?
• Traits are the result of Structure and Function
• Proteins determine structure and function
• DNA codes for proteins that confer traits
– DNA -> RNA -> Protein -> Trait
• Changes in DNA lead to proteins with:
– Different functions
– Novel traits
– Positive, negative, or no effects
• Genetic diversity provides pool for natural selection = evolution
Sample Preparation
Lab Period 1
Label one 1.5 ml fliptop tube for each of five fish samples.
Also label one screwcap micro tube for each fish
sample.
Add 250 μl of Bio-Rad Laemmli sample buffer to each
labeled fliptop microtube.
Cut a piece of each fish muscle about 0.25 x 0.25 x 0.25
cm3 and transfer each piece into a labeled fliptop tube.
(Close the lid!)
Sample Preparation
Lab Period 1 (con’t)
Flick the microtubes 15 times to agitate the tissue in the
sample buffer. Incubate for 5 minutes at room
temperature.
Carefully transfer the buffer by pouring from each fliptop
tube into a labeled screwcap tube. Do not transfer the
fish!
Heat the fish samples in screwcap microtubes for 5
minutes at 95°C. Freeze until lab period 2.
Electrophoresis
Lab Period 2
Heat extracted fish samples and actin and
myosin standard to 95°C for 2–5 min. This
dissolves any detergent in the extraction
(Laemmli) buffer that may have precipitated
upon freezing.
Electrophoresis
Lab Period 2 (con’t)
Load your gel:
• 5 μl Precision Plus Protein Kaleidoscope
prestained standards (Stds)
• 10 μl fish sample 1
• 10 μl fish sample 2
• 10 μl fish sample 3
• 10 μl actin and myosin standard (AM)
Electrophoresis
Lab Period 2 (con’t)
Electrophorese for 30 minutes at 200 V in 1x TGS
electrophoresis buffer.
After electrophoresis, remove gel from cassette
and transfer gel to a container with 25 ml BioSafe Coomassie blue stain per gel and stain gel
for 1 hour, with gentle shaking for best results.
Electrophoresis
Lab Period 3
Discard stain and destain gels in a large volume
of water for at least 30 minutes to overnight,
changing the water at least once. Bluestained bands will be visible on a clear gel
after destaining.
Dry gels using GelAir cellophane.
Analysis
Lab Period 3 (con’t)
Correlate bands of fish samples with AM and
Kaleidoscope standards.
Check online protein database for correlation of
sample proteins with those of the species in
the databases.
Guided by similarity of protein content, draw
cladogram relating fish species.